Method for detecting multispecific antibody light chain mispairing

ABSTRACT

Use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the analysis of the multispecific antibody&#39;s light chain pairing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/278,242, filed Sep. 28, 2016, which is a continuation ofInternational Patent Application No. PCT/EP2015/057164, having aninternational filing date of Apr. 1, 2015, the entire contents of whichare incorporated herein by reference, and which claims benefit under 35U.S.C. § 119 to European Patent Application No. 14163165.5, filed onApr. 2, 2014 and European Patent Application No. 15155361.7, filed onFeb. 17, 2015.

BACKGROUND OF THE INVENTION

Herein is reported a method for the determination of light chainmispairing in a multispecific antibody preparation based on limitedproteolytic digestion and ESI-MS analysis. The method can be used forquality control or cell line selection.

In 1995 Yamaguchi et al. (J. Immunol. Meth. 181 (1995) 259-267) reporteda method for proteolytic fragmentation with high specificity of mouseimmunoglobulin G using lysyl endopeptidase, clostripain,metalloendopeptidase and V8 protease, whereby under the reactionconditions examined clostripain, lysyl endopeptidase,metalloendopeptidase, and V8 protease failed to cleave IgG1 and IgG1-CMselectively in the hinge region.

Matsuda et al. (FEBS Lett. 473 (2000) 349-357) reported a method for thepreparation of Fc fragments using Lys-C for obtaining information of theFc-associated glycoforms by MS for determining the pairing ofoligosaccharides in the Fc region of immunoglobulin G.

Villanueva et al. (J. Mass Spectrom. 37 (2002) 974-984) reported alimited proteolysis methodology, based on the use of unspecificexoproteases coupled with matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS), to map quicklysecondary structure elements of a protein from both ends, the N- andC-termini.

In WO 2005/073732 a method of analysis of high molecular weightproteins, specifically, a reversed-phase LC/MS method of analysis ofhigh molecular weight proteins including antibodies, is reported.

WO 2006/047340 relates to subjecting column isolated preparations ofpolypeptides with a reduction/oxidation reagent and a chaotropic agentand isolating the refolded active protein produced from said contacting.The active proteins can be high molecular weight proteins including Fcdomain containing polypeptides or fragments.

Kleemann et al. (Anal. Chem. 80 (2008) 2001-2009) reported thecharacterization of IgG1 immunoglobulins and peptide-Fc fusion proteinsby limited proteolysis in conjunction with LC-MS. Applying limitedproteolysis using endoproteinase Lys-C resulted in the predominantcleavage C-terminal of this lysine residue.

SUMMARY OF THE INVENTION

It has been found that for the determination of light chain mispairingin a multispecific antibody limited proteolysis of the (multispecific)antibody has to be performed prior to MS analysis as the determinationof light chain mispairing in a complete multispecific antibody ishampered by isobaric mass interference.

Thus, one aspect as reported herein is the use of a limited digestionwith a proteolytic enzyme of a multispecific antibody for the analysisof the multispecific antibody's light chain pairing.

One aspect as reported herein is the use of a limited digestion with aproteolytic enzyme of a multispecific antibody for the determination oflight chain mispairing in the multispecific antibody.

One aspect as reported herein is the use of a limited digestion with aproteolytic enzyme of a multispecific antibody produced by a recombinantmammalian cell for the selection of a multispecific antibody producingmammalian cell.

In one embodiment of all previous aspects theanalysis/determination/selection is by using the result of a massspectrometry of the digested antibody.

One aspect as reported herein is a method for the determination of thelight chain pairing in a multispecific antibody comprising the followingsteps:

-   -   a) incubating a sample comprising the multispecific antibody        with a proteolytic enzyme for a limited time,    -   b) identifying the mass of the fragments obtained by the limited        proteolytic digestion in step a) by mass spectrometry, and    -   c) determining from the results in step b) the light chain        pairing of the multispecific antibody.

One aspect as reported herein is a method for the determination of lightchain mispairing of a multispecific antibody comprising the followingsteps:

-   -   a) incubating a sample comprising the multispecific antibody        with a proteolytic enzyme for a limited time,    -   b) identifying the mass of the fragments obtained by the limited        proteolytic digestion in step a) by mass spectrometry, and    -   c) determining from the results in step b) the light chain        pairing of the multispecific antibody and thereby determining        light chain mispairing.

One aspect as reported herein is a method for the selection of arecombinant mammalian cell producing a multispecific antibody comprisingthe following steps:

-   -   a) individually incubating a sample comprising a multispecific        antibody produced by a clonal population of a recombinant        mammalian cell of a multitude of recombinant mammalian cells all        cells of the multitude producing the same multispecific antibody        with a proteolytic enzyme for a limited time,    -   b) identifying the mass of the fragments obtained by the limited        proteolytic digestion in step a) by mass spectrometry,    -   c) determining from the results in step b) the presence of light        chain mispairing of the multispecific antibody for each clonal        cell population, and    -   d) selecting based on the results in step c) a recombinant cell        producing a multispecific antibody.        In one embodiment of all aspects the proteolytic enzyme is        selected from the group consisting of Lys-C, plasmin, Asp-N,        Arg-C, Glu-C, Ides, pepsin and chymotrypsin.

In one embodiment the multispecific antibody is a bivalent antibody.

In one preferred embodiment of all aspects the proteolytic enzyme isLys-C or plasmin.

In one embodiment the multispecific antibody is a tri- or tetravalentantibody. In one embodiment the multispecific antibody is a full lengthantibody comprising two light chains and two heavy chain whereby to theC-termini of the heavy chains a moiety independently of each otherselected from the group consisting of no moiety, an Fv fragment, a Fabfragment, a scFv fragment and a scFab fragment is conjugated eitherdirectly or via a peptidic linker.

In case the antibody is a tetravalent antibody wherein also bindingsites are attached to the C-termini of the heavy chains a doubledigestion with two proteolytic enzymes might be required.

In one embodiment the multispecific antibody is a trivalent ortetravalent multispecific antibody and the method comprises a doubledigestion by incubating with a mixture of two proteolytic enzymes.

In one embodiment the mixture of proteolytic enzymes is plasmin andIdes. In one embodiment the mixture of proteolytic enzymes is Lys-C andIdes.

In case of an effector silent antibody (L234A, L235A mutations) Idesdoes not cleave and pepsin has to be chosen.

In one embodiment the mixture of proteolytic enzymes is plasmin andpepsin. In one embodiment the mixture of proteolytic enzymes is Lys-Cand pepsin.

In one preferred embodiment the multivalent antibody is a bispecificantibody.

In one embodiment of all aspects the incubating is for up to 60 minutes.In one embodiment of all aspects the incubating is for 20 to 60 minutes.In one embodiment of all aspects the incubating is for 35 to 45 minutes.

In one preferred embodiment of all aspects the incubating is for about40 minutes.

In one embodiment of all aspects the multispecific antibody that isincubated with the proteolytic enzyme is a deglycosylated multispecificantibody.

In one embodiment of all aspects the weight ratio of antibody to enzymeis 1:150 to 1:500. In one embodiment of all aspects the weight ratio ofantibody to enzyme is 1:200 to 1:400. In one embodiment of all aspectsthe weight ratio of antibody to enzyme is about 1:200.

In one embodiment of all aspects the multispecific antibody that isincubated with the proteolytic enzyme has a concentration of from 200 to600 μg/mL.

In one embodiment of all aspects step b) is

-   -   b) desalting the incubation mixture of step a) and identifying        the mass of the fragments obtained by the limited proteolytic        digestion in step a) by mass spectrometry.

In one embodiment of all aspects the multispecific antibody is amonoclonal multispecific antibody.

In one embodiment of all aspects the multispecific antibody comprises atleast two non-peptidically associated light chains.

In one embodiment of all aspects the multispecific antibody comprises atleast three non-peptidically associated polypeptides.

In one embodiment of all aspects the mass spectrometry is an onlinedetermination.

In one embodiment of all aspects the mass spectrometry is an offlinedetermination. In one embodiment the offline mass spectrometry ispreceded by a desalting step.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a cell” includes a pluralityof such cells and equivalents thereof known to those skilled in the art,and so forth. As well, the terms “a” (or “an”), “one or more” and “atleast one” can be used interchangeably herein. In contrast theretoreference to “one cell” does not include a plurality of such cells butis limited to a single (isolated) cell.

It is also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Further it is also to be noted that the term “comprising” encompassesthe term “consisting of” as limitation.

The term “antibody” is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g., atleast bispecific antibodies) as long as these antibody structures aremultispecific and comprise at least two non-peptidically bound lightchains.

The term “non-peptidically bound” denotes that two polypeptides are notassociated with each other by a peptide bond (formed between amino- andcarboxy-groups of amino acids). Nevertheless these polypeptides can beassociated by other covalent bonds such as disulfide bonds ornon-covalently.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “Fc-region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc-regions andvariant Fc-regions. In one embodiment, a human IgG heavy chain Fc-regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc-regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc-region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242.

The terms “full length antibody”, “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt,T. J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y.(2007), page 91) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991)624-628).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a method for the determination of light chainmispairing in a multispecific antibody preparation based on limitedproteolytic digestion and ESI-MS analysis. The method can be used forquality control or cell line selection.

It has been found that for the determination of light chain mispairingin a multispecific antibody limited proteolysis of the (multispecific)antibody has to be performed prior to MS analysis as the determinationof light chain mispairing in a complete multispecific antibody ishampered by isobaric mass interference.

Thus, one aspect as reported herein is the use of a limited digestionwith a proteolytic enzyme of a multispecific antibody for the analysisof the multispecific antibody's light chain pairing.

In a bivalent multispecific antibody, i.e. in a bivalent bispecificantibody, one light chain mispairing product is possible (see FIG. 1).

In one exemplary embodiment of the methods as reported herein twosamples of a bivalent bispecific antibody has been digested with plasmin(EC 3.4.21.7). In all samples no uncleaved intact antibody was presentafter the digestion. In sample 1 approximately 20% of light-chainmispaired antibody is detectable (see FIG. 3) whereas in the secondsample no light-chain mispaired antibody is detectable (see FIG. 4).

In one exemplary embodiment of the methods as reported herein a sampleof a bivalent bispecific antibody has been limited digested with Lys-C.In the sample light-chain mispaired antibody is detectable (see FIG. 5).

If more than 2 light chains are present, e.g. in a trivalent ortetravalent multispecific antibody, more than one light chain mispairingproduct is possible (see FIG. 2). In a mass spectrometric analysis ofthe complete antibody it is only possible to determine the presence of alight chain mispaired product but not the stoichiometry and theorientation of a potential mispairing cannot be detected (see FIG. 6).

Different proteolytic enzymes can be chosen for this task:

-   -   Ides: cleaves below the hinge region (cannot be used in an        antibody with the L234A, L235A mutation)    -   plasmin: cleaves above the hinge region    -   pepsin: cleaves below the hinge region; results in a degraded        Fc-region    -   Lys-C: cleaves above the hinge region.

In one exemplary embodiment of the methods as reported herein a sampleof a tetravalent bispecific antibody has been limited digested withLys-C.

It has been found that for a tetravalent multispecific antibody themispairing in the Fab region form from light chain 1 and the heavy chain1 fragment can be detected better at an ISCID of 0 whereas the Fc-regionfragment comprising the second light chain can be detected better at anISCID of 90.

In order to remove the Fc-region a Protein A affinity chromatography canbe used to separate the Fab fragments from the Fc-region after theenzymatic digestion. The Fab fragments are in the flow-through of theProtein A affinity chromatography.

Chimeric and Humanized Antibodies

In certain embodiments, an antibody analyzed with the methods asreported herein is a chimeric antibody. Certain chimeric antibodies aredescribed, e.g., in U.S. Pat. No. 4,816,567; and Morrison, S. L. et al.,Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, achimeric antibody comprises a non-human variable region (e.g., avariable region derived from a mouse, rat, hamster, rabbit, or non-humanprimate, such as a monkey) and a human constant region. In a furtherexample, a chimeric antibody is a “class switched” antibody in which theclass or subclass has been changed from that of the parent antibody.Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I. et al., Nature 332 (1988)323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describingspecificity determining region (SDR) grafting); Padlan, E. A., Mol.Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, W. F.et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); andOsbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br.J. Cancer 83 (2000) 252-260 (describing the “guided selection” approachto FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308;framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89(1992) 4285-4289; and Presta, L. G. et al., J. Immunol. 151 (1993)2623-2632); human mature (somatically mutated) framework regions orhuman germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

Human Antibodies

In certain embodiments, an antibody analyzed in the method as reportedherein is a human antibody. Human antibodies can be produced usingvarious techniques known in the art. Human antibodies are describedgenerally in van Dijk, M. A. and van de Winkel, J. G., Curr. Opin.Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20(2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMab®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and US 2007/0061900, describing VelociMouse® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boemer, P. et al., J. Immunol. 147(1991) 86-95) Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J. et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Library-Derived Antibodies

Antibodies analyzed in the methods as reported herein may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. For example, a variety of methods are known inthe art for generating phage display libraries and screening suchlibraries for antibodies possessing the desired binding characteristics.Such methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods inMolecular Biology 178 (2001) 1-37 and further described, e.g., in theMcCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al.,Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol. Biol. 222 (1992)581-597; Marks, J. D. and Bradbury, A., Methods in Molecular Biology 248(2003) 161-175; Sidhu, S. S. et al., J. Mol. Biol. 338 (2004) 299-310;Lee, C. V. et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, F. A.,Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, C. V. etal., J. Immunol. Methods 284 (2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G. et al., Ann. Rev.Immunol. 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self antigens without any immunization as described by Griffiths,A. D. et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and US2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

Multispecific Antibodies

Engineered proteins, such as bi- or multispecific antibodies capable ofbinding two or more antigens are known in the art. Such multispecificbinding proteins can be generated using cell fusion, chemicalconjugation, or recombinant DNA techniques.

In certain embodiments, an antibody analyzed in the methods as reportedherein is a multispecific antibody, e.g. at least a bispecific antibody.Multispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different sites. Multispecific antibodiescan be prepared as full length antibodies.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (sFv) dimers (see, e.g. Gruber, M et al., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” comprising an antigen binding site that binds to [[PRO]] as wellas another, different antigen (see, US 2008/0069820, for example).

The antibody or fragment herein also includes multispecific antibodiesdescribed in

WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO2010/145793.

A wide variety of recombinant multispecific antibody formats have beendeveloped in the recent past, e.g. tetravalent bispecific antibodies byfusion of, e.g. an IgG antibody format and single chain domains (seee.g. Coloma, M. J., et. al., Nature Biotech. 15 (1997) 159-163; WO2001/077342; and Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234.

Also several other new formats wherein the antibody core structure (IgA,IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- ortetrabodies, minibodies, several single chain formats (scFv, Bis-scFv),which are capable of binding two or more antigens, have been developed(Holliger, P., et. al, Nature Biotech. 23 (2005) 1126-1136; Fischer, N.,and Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et. al., J.Immunol. Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech. 25(2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD,IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fusee.g. two Fab fragments or scFv (Fischer, N., and Léger, O., Pathobiology74 (2007) 3-14). While it is obvious that linkers have advantages forthe engineering of multispecific antibodies, they may also causeproblems in therapeutic settings. Indeed, these foreign peptides mightelicit an immune response against the linker itself or the junctionbetween the protein and the linker. Furthermore, the flexible nature ofthese peptides makes them more prone to proteolytic cleavage,potentially leading to poor antibody stability, aggregation andincreased immunogenicity. In addition one may want to retain effectorfunctions, such as e.g. complement-dependent cytotoxicity (CDC) orantibody dependent cellular cytotoxicity (ADCC), which are mediatedthrough the Fc-part by maintaining a high degree of similarity tonaturally occurring antibodies.

Thus, ideally, one should aim at developing multispecific antibodiesthat are very similar in general structure to naturally occurringantibodies (like IgA, IgD, IgE, IgG or IgM) with minimal deviation fromhuman sequences.

In one approach bispecific antibodies that are very similar to naturalantibodies have been produced using the quadroma technology (seeMilstein, C., and Cuello, A. C., Nature 305 (1983) 537-540) based on thesomatic fusion of two different hybridoma cell lines expressing murinemonoclonal antibodies with the desired specificities of the bispecificantibody. Because of the random pairing of two different antibody heavyand light chains within the resulting hybrid-hybridoma (or quadroma)cell line, up to ten different antibody species are generated of whichonly one is the desired, functional bispecific antibody. Due to thepresence of mispaired byproducts, and significantly reduced productionyields, sophisticated purification procedures are required (see e.g.Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234). In general thesame problem of mispaired by-products remains if recombinant expressiontechniques are used.

An approach to circumvent the problem of mispaired byproducts, which isknown as ‘knobs-into-holes’, aims at forcing the pairing of twodifferent antibody heavy chains by introducing mutations into the CH3domains to modify the contact interface. On one chain bulky amino acidswere replaced by amino acids with short side chains to create a ‘hole’.Conversely, amino acids with large side chains were introduced into theother CH3 domain, to create a ‘knob’. By co-expressing these two heavychains (and two identical light chains, which have to be appropriate forboth heavy chains), high yields of heterodimer formation (‘knob-hole’)versus homodimer formation (‘hole-hole’ or ‘knob-knob’) was observed(Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; and WO96/027011). The percentage of heterodimer could be further increased byremodeling the interaction surfaces of the two CH3 domains using a phagedisplay approach and the introduction of a disulfide bridge to stabilizethe heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998)677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). Newapproaches for the knobs-into-holes technology are described in e.g. inEP 1 870 459 A1. Although this format appears very attractive, no datadescribing progression towards the clinic are currently available. Oneimportant constraint of this strategy is that the light chains of thetwo parent antibodies have to be identical to prevent mispairing andformation of inactive molecules. Thus this technique is not appropriateas a basis for easily developing recombinant, tri- or tetraspecificantibodies against three or four antigens starting from two antibodiesagainst the first and the second antigen, as either the heavy chains ofthese antibodies and/or the identical light chains have to be optimizedfirst and then further antigen binding peptides against the third andfourth antigen have to be added.

WO 2006/093794 relates to heterodimeric protein binding compositions. WO99/37791 describes multipurpose antibody derivatives. Morrison, S. L.,et al., J. Immunol. 160 (1998) 2802-2808 refers to the influence ofvariable region domain exchange on the functional properties of IgG.

WO 2013/02362 relates to heterodimerized polypeptides. WO 2013/12733relates to polypeptides comprising heterodimeric Fc regions. WO2012/131555 relates to engineered hetero-dimeric immunoglobulins. EP2647707 relates to engineered hetero-dimeric immunoglobulins.

WO 2013/026835 relates to bispecific, Fc free antibodies with a domaincrossover. WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191 relateto bivalent, bispecific IgG antibodies with a domain crossover.

The multispecific antibodies with VH/VL replacement/exchange in onebinding to prevent light chain mispairing (CrossMabVH-VL) which aredescribed in WO2009/080252, (see also Schaefer, W. et al, PNAS, 108(2011) 11187-1191) clearly reduce the byproducts caused by the mismatchof a light chain against a first antigen with the wrong heavy chainagainst the second antigen (compared to approaches without such domainexchange). However their preparation is not completely free of sideproducts. The main side product is based on a Bence-Jones-typeinteraction (see also Schaefer, W. et al, PNAS, 108 (2011) 11187-1191;in Fig. S1I of the Supplement).

Bispecific Antibodies

In one embodiment the multispecific antibody is a bispecific antibody.

One aspect as reported herein is a bivalent, bispecific antibodycomprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the variable        domains VL and VH of the second light chain and the second heavy        chain are replaced by each other,    -   wherein the first and the second antigen are different antigens.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.

In the antibody under b)

within the light chain

-   -   the variable light chain domain VL is replaced by the variable        heavy chain domain VH of said antibody,

and

within the heavy chain

-   -   the variable heavy chain domain VH is replaced by the variable        light chain domain VL of said antibody.

In one embodiment

-   -   i) in the constant domain CL of the first light chain under a)        the amino acid at position 124 (numbering according to Kabat) is        substituted by a positively charged amino acid, and wherein in        the constant domain CH1 of the first heavy chain under a) the        amino acid at position 147 or the amino acid at position 213        (numbering according to Kabat EU index) is substituted by a        negatively charged amino acid,    -   or    -   ii) in the constant domain CL of the second light chain under b)        the amino acid at position 124 (numbering according to Kabat) is        substituted by a positively charged amino acid, and wherein in        the constant domain CH1 of the second heavy chain under b) the        amino acid at position 147 or the amino acid at position 213        (numbering according to Kabat EU index) is substituted by a        negatively charged amino acid.

In one preferred embodiment

-   -   i) in the constant domain CL of the first light chain under a)        the amino acid at position 124 is substituted independently by        lysine (K), arginine (R) or histidine (H) (numbering according        to Kabat) (in one preferred embodiment independently by        lysine (K) or arginine (R)), and wherein in the constant domain        CH1 of the first heavy chain under a) the amino acid at position        147 or the amino acid at position 213 is substituted        independently by glutamic acid (E) or aspartic acid (D)        (numbering according to Kabat EU index),    -   or    -   ii) in the constant domain CL of the second light chain under b)        the amino acid at position 124 is substituted independently by        lysine (K), arginine (R) or histidine (H) (numbering according        to Kabat) (in one preferred embodiment independently by        lysine (K) or arginine (R)), and wherein in the constant domain        CH1 of the second heavy chain under b) the amino acid at        position 147 or the amino acid at position 213 is substituted        independently by glutamic acid (E) or aspartic acid (D)        (numbering according to Kabat EU index).

In one embodiment in the constant domain CL of the second heavy chainthe amino acids at position 124 and 123 are substituted by K (numberingaccording to Kabat EU index).

In one embodiment in the constant domain CH1 of the second light chainthe amino acids at position 147 and 213 are substituted by E (numberingaccording to EU index of Kabat).

In one preferred embodiment in the constant domain CL of the first lightchain the amino acids at position 124 and 123 are substituted by K, andin the constant domain CH1 of the first heavy chain the amino acids atposition 147 and 213 are substituted by E (numbering according to KabatEU index).

In one embodiment in the constant domain CL of the second heavy chainthe amino acids at position 124 and 123 are substituted by K, andwherein in the constant domain CH1 of the second light chain the aminoacids at position 147 and 213 are substituted by E, and in the variabledomain VL of the first light chain the amino acid at position 38 issubstituted by K, in the variable domain VH of the first heavy chain theamino acid at position 39 is substituted by E, in the variable domain VLof the second heavy chain the amino acid at position 38 is substitutedby K, and in the variable domain VH of the second light chain the aminoacid at position 39 is substituted by E (numbering according to Kabat EUindex).

One aspect as reported herein is a bivalent, bispecific antibodycomprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the variable        domains VL and VH of the second light chain and the second heavy        chain are replaced by each other, and wherein the constant        domains CL and CH1 of the second light chain and the second        heavy chain are replaced by each other,    -   wherein the first and the second antigen are different antigens.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain and a) are isolated chains.

In the antibody under b)

within the light chain

-   -   the variable light chain domain VL is replaced by the variable        heavy chain domain VH of said antibody, and the constant light        chain domain CL is replaced by the constant heavy chain domain        CH1 of said antibody;

and

within the heavy chain

-   -   the variable heavy chain domain VH is replaced by the variable        light chain domain VL of said antibody, and the constant heavy        chain domain CH1 is replaced by the constant light chain domain        CL of said antibody.

One aspect as reported herein is a bivalent, bispecific antibodycomprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the constant        domains CL and CH1 of the second light chain and the second        heavy chain are replaced by each other,    -   wherein the first and the second antigen are different antigens.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.

In the antibody under b)

within the light chain

-   -   the constant light chain domain CL is replaced by the constant        heavy chain domain CH1 of said antibody;

and within the heavy chain

-   -   the constant heavy chain domain CH1 is replaced by the constant        light chain domain CL of said antibody.

One aspect as reported herein is a multispecific antibody comprising

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) one, two, three or four single chain Fab fragments        specifically binding to one to four further antigens (i.e. a        second and/or third and/or fourth and/or fifth antigen,        preferably specifically binding to one further antigen, i.e. a        second antigen),    -   wherein said single chain Fab fragments under b) are fused to        said full length antibody under a) via a peptidic linker at the        C- or N-terminus of the heavy or light chain of said full length        antibody,    -   wherein the first and the second antigen are different antigens.

In one embodiment one or two identical single chain Fab fragmentsbinding to a second antigen are fused to said full length antibody via apeptidic linker at the C-terminus of the heavy or light chains of saidfull length antibody.

In one embodiment one or two identical single chain Fab fragmentsbinding to a second antigen are fused to said full length antibody via apeptidic linker at the C-terminus of the heavy chains of said fulllength antibody.

In one embodiment one or two identical single chain Fab fragmentsbinding to a second antigen are fused to said full length antibody via apeptidic linker at the C-terminus of the light chains of said fulllength antibody.

In one embodiment two identical single chain Fab fragments binding to asecond antigen are fused to said full length antibody via a peptidiclinker at the C-terminus of each heavy or light chain of said fulllength antibody.

In one embodiment two identical single chain Fab fragments binding to asecond antigen are fused to said full length antibody via a peptidiclinker at the C-terminus of each heavy chain of said full lengthantibody.

In one embodiment two identical single chain Fab fragments binding to asecond antigen are fused to said full length antibody via a peptidiclinker at the C-terminus of each light chain of said full lengthantibody.

One aspect as reported herein is a trivalent, bispecific antibodycomprising

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains,    -   b) a first polypeptide consisting of        -   ba) an antibody heavy chain variable domain (VH),        -   or        -   bb) an antibody heavy chain variable domain (VH) and an            antibody constant domain 1 (CH1),    -    wherein said first polypeptide is fused with the N-terminus of        its VH domain via a peptidic linker to the C-terminus of one of        the two heavy chains of said full length antibody,    -   c) a second polypeptide consisting of        -   ca) an antibody light chain variable domain (VL),        -   or        -   cb) an antibody light chain variable domain (VL) and an            antibody light chain constant domain (CL),    -    wherein said second polypeptide is fused with the N-terminus of        the VL domain via a peptidic linker to the C-terminus of the        other of the two heavy chains of said full length antibody,    -   and    -   wherein the antibody heavy chain variable domain (VH) of the        first polypeptide and the antibody light chain variable domain        (VL) of the second polypeptide together form an antigen-binding        site specifically binding to a second antigen,    -   and    -   wherein the first and the second antigen are different antigens.

In one embodiment the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) are linked and stabilized via an interchaindisulfide bridge by introduction of a disulfide bond between thefollowing positions:

-   -   i) heavy chain variable domain position 44 to light chain        variable domain position 100, or    -   ii) heavy chain variable domain position 105 to light chain        variable domain position 43, or    -   iii) heavy chain variable domain position 101 to light chain        variable domain position 100 (numbering always according to        Kabat EU index).

Techniques to introduce unnatural disulfide bridges for stabilizationare described e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Eng.(1997) 1453-59; Kobayashi, H., et al., Nuclear Medicine & Biology, Vol.25, (1998) 387-393; or Schmidt, M., et al., Oncogene (1999) 181711-1721. In one embodiment the optional disulfide bond between thevariable domains of the polypeptides under b) and c) is between heavychain variable domain position 44 and light chain variable domainposition 100. In one embodiment the optional disulfide bond between thevariable domains of the polypeptides under b) and c) is between heavychain variable domain position 105 and light chain variable domainposition 43. (numbering always according to EU index of Kabat) In oneembodiment a trivalent, bispecific antibody without said optionaldisulfide stabilization between the variable domains VH and VL of thesingle chain Fab fragments is preferred.

One aspect as reported herein is a trispecific or tetraspecificantibody, comprising

-   -   a) a first light chain and a first heavy chain of a full length        antibody which specifically binds to a first antigen, and    -   b) a second (modified) light chain and a second (modified) heavy        chain of a full length antibody which specifically binds to a        second antigen, wherein the variable domains VL and VH are        replaced by each other, and/or wherein the constant domains CL        and CH1 are replaced by each other, and    -   c) wherein one to four antigen binding peptides which        specifically bind to one or two further antigens (i.e. to a        third and/or fourth antigen) are fused via a peptidic linker to        the C- or N-terminus of the light chains or heavy chains of a)        and/or b), wherein the first and the second antigen are        different antigens.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain and a) are isolated chains.

In one embodiment the trispecific or tetraspecific antibody comprisesunder c) one or two antigen binding peptides which specifically bind toone or two further antigens.

In one embodiment the antigen binding peptides are selected from thegroup of a scFv fragment and a scFab fragment.

In one embodiment the antigen binding peptides are scFv fragments.

In one embodiment the antigen binding peptides are scFab fragments.

In one embodiment the antigen binding peptides are fused to theC-terminus of the heavy chains of a) and/or b).

In one embodiment the trispecific or tetraspecific antibody comprisesunder c) one or two antigen binding peptides which specifically bind toone further antigen.

In one embodiment the trispecific or tetraspecific antibody comprisesunder c) two identical antigen binding peptides which specifically bindto a third antigen. In one preferred embodiment such two identicalantigen binding peptides are fused both via the same peptidic linker tothe C-terminus of the heavy chains of a) and b). In one preferredembodiment the two identical antigen binding peptides are either a scFvfragment or a scFab fragment.

In one embodiment the trispecific or tetraspecific antibody comprisesunder c) two antigen binding peptides which specifically bind to a thirdand a fourth antigen. In one embodiment said two antigen bindingpeptides are fused both via the same peptide connector to the C-terminusof the heavy chains of a) and b). In one preferred embodiment said twoantigen binding peptides are either a scFv fragment or a scFab fragment.

One aspect as reported herein is a bispecific, tetravalent antibodycomprising

-   -   a) two light chains and two heavy chains of an antibody, which        specifically bind to a first antigen (and comprise two Fab        fragments),    -   b) two additional Fab fragments of an antibody, which        specifically bind to a second antigen, wherein said additional        Fab fragments are fused both via a peptidic linker either to the        C- or N-termini of the heavy chains of a),    -   and    -   wherein in the Fab fragments the following modifications were        performed        -   i) in both Fab fragments of a), or in both Fab fragments of            b), the variable domains VL and VH are replaced by each            other, and/or the constant domains CL and CH1 are replaced            by each other,        -   or        -   ii) in both Fab fragments of a) the variable domains VL and            VH are replaced by each other, and the constant domains CL            and CH1 are replaced by each other,            -   and            -   in both Fab fragments of b) the variable domains VL and                VH are replaced by each other, or the constant domains                CL and CH1 are replaced by each other,        -   or        -   iii) in both Fab fragments of a) the variable domains VL and            VH are replaced by each other, or the constant domains CL            and CH1 are replaced by each other,            -   and            -   in both Fab fragments of b) the variable domains VL and                VH are replaced by each other, and the constant domains                CL and CH1 are replaced by each other,        -   or        -   iv) in both Fab fragments of a) the variable domains VL and            VH are replaced by each other, and in both Fab fragments            of b) the constant domains CL and CH1 are replaced by each            other,        -   or        -   v) in both Fab fragments of a) the constant domains CL and            CH1 are replaced by each other, and in both Fab fragments            of b) the variable domains VL and VH are replaced by each            other,    -   wherein the first and the second antigen are different antigens.

In one embodiment said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a), or tothe N-termini of the heavy chains of a).

In one embodiment said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a).

In one embodiment said additional Fab fragments are fused both via apeptide connector to the N-termini of the heavy chains of a).

In one embodiment in the Fab fragments the following modifications areperformed:

-   -   i) in both Fab fragments of a), or in both Fab fragments of b),        the variable domains VL and VH are replaced by each other,        -   and/or        -   the constant domains CL and CH1 are replaced by each other.

In one embodiment in the Fab fragments the following modifications areperformed:

-   -   i) in both Fab fragments of a) the variable domains VL and VH        are replaced by each other,        -   and/or        -   the constant domains CL and CH1 are replaced by each other.

In one embodiment in the Fab fragments the following modifications areperformed:

-   -   i) in both Fab fragments of a) the constant domains CL and CH1        are replaced by each other.

In one embodiment in the Fab fragments the following modifications areperformed:

-   -   i) in both Fab fragments of b) the variable domains VL and VH        are replaced by each other,        -   and/or        -   the constant domains CL and CH1 are replaced by each other.

In one embodiment in the Fab fragments the following modifications areperformed:

-   -   i) in both Fab fragments of b) the constant domains CL and CH1        are replaced by each other.

One aspect as reported herein is a bispecific, tetravalent antibodycomprising:

-   -   a) a (modified) heavy chain of a first antibody, which        specifically binds to a first antigen and comprises a first        VH-CH1 domain pair, wherein to the C-terminus of said heavy        chain the N-terminus of a second VH-CH1 domain pair of said        first antibody is fused via a peptidic linker,    -   b) two light chains of said first antibody of a),    -   c) a (modified) heavy chain of a second antibody, which        specifically binds to a second antigen and comprises a first        VH-CL domain pair, wherein to the C-terminus of said heavy chain        the N-terminus of a second VH-CL domain pair of said second        antibody is fused via a peptidic linker, and    -   d) two (modified) light chains of said second antibody of c),        each comprising a CL-CH1 domain pair,    -   wherein the first and the second antigen are different antigens.

One aspect as reported herein is a bispecific antibody comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to a first antigen, and    -   b) the heavy chain and the light chain of a second full length        antibody that specifically binds to a second antigen, wherein        the N-terminus of the heavy chain is connected to the C-terminus        of the light chain via a peptidic linker,    -   wherein the first and the second antigen are different.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain are isolated chains.

One aspect as reported herein is a bispecific antibody comprising

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) an Fv fragment specifically binding to a second antigen        comprising a VH² domain and a VL² domain, wherein both domains        are connected to each other via a disulfide bridge,    -   wherein only either the VH² domain or the VL² domain is fused        via a peptidic linker to the heavy or light chain of the full        length antibody specifically binding to a first antigen,    -   wherein the first and the second antigen are different antigens.

In the bispecific the heavy chains and the light chains under a) areisolated chains.

In one embodiment the other of the VH² domain or the VL² domain is notfused via a peptide linker to the heavy or light chain of the fulllength antibody specifically binding to a first antigen.

In all aspects as reported herein the first light chain comprises a VLdomain and a CL domain and the first heavy chain comprises a VH domain,a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

In one embodiment of all aspects the antibody as reported herein is amultispecific antibody, which requires heterodimerization of at leasttwo heavy chain polypeptides.

Several approaches for CH3-modifications in order to supportheterodimerization have been described, for example in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO2013/157954, WO 2013/096291, which are herein included by reference.Typically, in the approaches known in the art, the CH3 domain of thefirst heavy chain and the CH3 domain of the second heavy chain are bothengineered in a complementary manner so that the heavy chain comprisingone engineered CH3 domain can no longer homodimerize with another heavychain of the same structure (e.g. a CH3-engineered first heavy chain canno longer homodimerize with another CH3-engineered first heavy chain;and a CH3-engineered second heavy chain can no longer homodimerize withanother CH3-engineered second heavy chain). Thereby the heavy chaincomprising one engineered CH3 domain is forced to heterodimerize withanother heavy chain comprising the CH3 domain, which is engineered in acomplementary manner. For this embodiment of the invention, the CH3domain of the first heavy chain and the CH3 domain of the second heavychain are engineered in a complementary manner by amino acidsubstitutions, such that the first heavy chain and the second heavychain are forced to heterodimerize, whereas the first heavy chain andthe second heavy chain can no longer homodimerize (e.g. for stericreasons).

The different approaches for supporting heavy chain heterodimerizationknown in the art, that were cited and included above, are contemplatedas different alternatives used in a multispecific antibody according tothe invention, which comprises a “non-crossed Fab region” derived from afirst antibody, which specifically binds to a first antigen, and a“crossed Fab region” derived from a second antibody, which specificallybinds to a second antigen, in combination with the particular amino acidsubstitutions described above for the invention.

The CH3 domains of the multispecific antibody as reported herein can bealtered by the “knob-into-holes” technology which is described in detailwith several examples in e.g. WO 96/027011, Ridgway, J. B., et al.,Protein Eng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat.Biotechnol. 16 (1998) 677-681. In this method the interaction surfacesof the two CH3 domains are altered to increase the heterodimerization ofboth heavy chains containing these two CH3 domains. Each of the two CH3domains (of the two heavy chains) can be the “knob”, while the other isthe “hole”. The introduction of a disulfide bridge further stabilizesthe heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998)677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) andincreases the yield.

In one preferred embodiment the multispecific antibody as reportedherein comprises a T366W mutation in the CH3 domain of the “knobs chain”and T366S, L368A, Y407V mutations in the CH3 domain of the “hole-chain”(numbering according to Kabat EU index). An additional interchaindisulfide bridge between the CH3 domains can also be used (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681) e.g. by introducing aY349C mutation into the CH3 domain of the “knobs chain” and a E356Cmutation or a S354C mutation into the CH3 domain of the “hole chain”.Thus in a another preferred embodiment, the multispecific antibody asreported herein comprises the Y349C and T366W mutations in one of thetwo CH3 domains and the E356C, T366S, L368A and Y407V mutations in theother of the two CH3 domains or the multispecific antibody as reportedherein comprises the Y349C and T366W mutations in one of the two CH3domains and the S354C, T366S, L368A and Y407V mutations in the other ofthe two CH3 domains (the additional Y349C mutation in one CH3 domain andthe additional E356C or S354C mutation in the other CH3 domain forming ainterchain disulfide bridge) (numbering according to Kabat EU index).

But also other knobs-in-holes technologies as described by EP 1 870459A1, can be used alternatively or additionally. In one embodiment themultispecific antibody as reported herein comprises the R409D and K370Emutations in the CH3 domain of the “knobs chain” and the D399K and E357Kmutations in the CH3 domain of the “hole-chain” (numbering according toKabat EU index).

In one embodiment the multispecific antibody as reported hereincomprises a T366W mutation in the CH3 domain of the “knobs chain” andthe T366S, L368A and Y407V mutations in the CH3 domain of the “holechain” and additionally the R409D and K370E mutations in the CH3 domainof the “knobs chain” and the D399K and E357K mutations in the CH3 domainof the “hole chain” (numbering according to the Kabat EU index).

In one embodiment the multispecific antibody as reported hereincomprises the Y349C and T366W mutations in one of the two CH3 domainsand the S354C, T366S, L368A and Y407V mutations in the other of the twoCH3 domains, or the multispecific antibody as reported herein comprisesthe Y349C and T366W mutations in one of the two CH3 domains and theS354C, T366S, L368A and Y407V mutations in the other of the two CH3domains and additionally the R409D and K370E mutations in the CH3 domainof the “knobs chain” and the D399K and E357K mutations in the CH3 domainof the “hole chain” (numbering according to the Kabat EU index).

Apart from the “knob-into-hole technology” other techniques formodifying the CH3 domains of the heavy chains of a multispecificantibody to enforce heterodimerization are known in the art. Thesetechnologies, especially the ones described in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO2013/157954 and WO 2013/096291 are contemplated herein as alternativesto the “knob-into-hole technology” in combination with a multispecificantibody as reported herein.

In one embodiment of a multispecific antibody as reported herein theapproach described in EP 1870459 is used to support heterodimerizationof the first heavy chain and the second heavy chain of the multispecificantibody. This approach is based on the introduction of charged aminoacids with opposite charges at specific amino acid positions in theCH3/CH3-domain-interface between both, the first and the second heavychain.

Accordingly, this embodiment relates to a multispecific antibody asreported herein, wherein in the tertiary structure of the antibody theCH3 domain of the first heavy chain and the CH3 domain of the secondheavy chain form an interface that is located between the respectiveantibody CH3 domains, wherein the respective amino acid sequences of theCH3 domain of the first heavy chain and the CH3 domain of the secondheavy chain each comprise a set of amino acids that is located withinsaid interface in the tertiary structure of the antibody, wherein fromthe set of amino acids that is located in the interface in the CH3domain of one heavy chain a first amino acid is substituted by apositively charged amino acid and from the set of amino acids that islocated in the interface in the CH3 domain of the other heavy chain asecond amino acid is substituted by a negatively charged amino acid. Themultispecific antibody according to this embodiment is herein alsoreferred to as “CH3(+/−)-engineered multispecific antibody” (wherein theabbreviation “+/−” stands for the oppositely charged amino acids thatwere introduced in the respective CH3 domains).

In one embodiment of said CH3(+/−)-engineered multispecific antibody asreported herein the positively charged amino acid is selected from K, Rand H, and the negatively charged amino acid is selected from E or D.

In one embodiment of said CH3(+/−)-engineered multispecific antibody asreported herein the positively charged amino acid is selected from K andR, and the negatively charged amino acid is selected from E or D.

In one embodiment of said CH3(+/−)-engineered multispecific antibody asreported herein the positively charged amino acid is K, and thenegatively charged amino acid is E.

In one embodiment of said CH3(+/−)-engineered multispecific antibody asreported herein in the CH3 domain of one heavy chain the amino acid R atposition 409 is substituted by D and the amino acid K at position issubstituted by E, and in the CH3 domain of the other heavy chain theamino acid D at position 399 is substituted by K and the amino acid E atposition 357 is substituted by K (numbering according to Kabat EUindex).

In one embodiment of a multispecific antibody as reported herein theapproach described in WO2013/157953 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said multispecificantibody as reported herein, in the CH3 domain of one heavy chain theamino acid T at position 366 is substituted by K, and in the CH3 domainof the other heavy chain the amino acid L at position 351 is substitutedby D (numbering according to Kabat EU index). In another embodiment ofsaid multispecific antibody as reported herein, in the CH3 domain of oneheavy chain the amino acid T at position 366 is substituted by K and theamino acid L at position 351 is substituted by K, and in the CH3 domainof the other heavy chain the amino acid L at position 351 is substitutedby D (numbering according to Kabat EU index).

In another embodiment of said multispecific antibody as reported herein,in the CH3 domain of one heavy chain the amino acid T at position 366 issubstituted by K and the amino acid L at position 351 is substituted byK, and in the CH3 domain of the other heavy chain the amino acid L atposition 351 is substituted by D (numbering according to Kabat EUindex). Additionally at least one of the following substitutions iscomprised in the CH3 domain of the other heavy chain: the amino acid Yat position 349 is substituted by E, the amino acid Y at position 349 issubstituted by D and the amino acid L at position 368 is substituted byE (numbering according to Kabat EU index). In one embodiment the aminoacid L at position 368 is substituted by E (numbering according to KabatEU index).

In one embodiment of a multispecific antibody as reported herein theapproach described in WO2012/058768 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said multispecificantibody as reported herein, in the CH3 domain of one heavy chain theamino acid L at position 351 is substituted by Y and the amino acid Y atposition 407 is substituted by A, and in the CH3 domain of the otherheavy chain the amino acid T at position 366 is substituted by A and theamino acid K at position 409 is substituted by F (numbering according toKabat EU index). In another embodiment, in addition to theaforementioned substitutions, in the CH3 domain of the other heavy chainat least one of the amino acids at positions 411 (originally T), 399(originally D), 400 (originally S), 405 (originally F), 390 (originallyN) and 392 (originally K) is substituted (numbering according to KabatEU index). Preferred substitutions are:

-   -   substituting the amino acid T at position 411 by an amino acid        selected from N, R, Q, K, D, E and W (numbering according to        Kabat EU index),    -   substituting the amino acid D at position 399 by an amino acid        selected from R, W, Y, and K (numbering according to Kabat EU        index),    -   substituting the amino acid S at position 400 by an amino acid        selected from E, D, R and K (numbering according to Kabat EU        index),    -   substituting the amino acid F at position 405 by an amino acid        selected from I, M, T, S, V and W (numbering according to Kabat        EU index;    -   substituting the amino acid N at position 390 by an amino acid        selected from R, K and D (numbering according to Kabat EU index;        and    -   substituting the amino acid K at position 392 by an amino acid        selected from V, M, R, L, F and E (numbering according to Kabat        EU index).

In another embodiment of said multispecific antibody as reported herein(engineered according to WO2012/058768), in the CH3 domain of one heavychain the amino acid L at position 351 is substituted by Y and the aminoacid Y at position 407 is substituted by A, and in the CH3 domain of theother heavy chain the amino acid T at position 366 is substituted by Vand the amino acid K at position 409 is substituted by F (numberingaccording to Kabat EU index). In another embodiment of saidmultispecific antibody as reported herein, in the CH3 domain of oneheavy chain the amino acid Y at position 407 is substituted by A, and inthe CH3 domain of the other heavy chain the amino acid T at position 366is substituted by A and the amino acid K at position 409 is substitutedby F (numbering according to Kabat EU index). In said lastaforementioned embodiment, in the CH3 domain of said other heavy chainthe amino acid K at position 392 is substituted by E, the amino acid Tat position 411 is substituted by E, the amino acid D at position 399 issubstituted by R and the amino acid S at position 400 is substituted byR (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein theapproach described in WO 2011/143545 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said multispecificantibody as reported herein, amino acid modifications in the CH3 domainsof both heavy chains are introduced at positions 368 and/or 409(numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein theapproach described in WO 2011/090762 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. WO 2011/090762 relates to amino acidmodifications according to the “knob-into-hole” technology. In oneembodiment of said CH3(KiH)-engineered multispecific antibody asreported herein, in the CH3 domain of one heavy chain the amino acid Tat position 366 is substituted by W, and in the CH3 domain of the otherheavy chain the amino acid Y at position 407 is substituted by A(numbering according to Kabat EU index). In another embodiment of saidCH3(KiH)-engineered multispecific antibody as reported herein, in theCH3 domain of one heavy chain the amino acid T at position 366 issubstituted by Y, and in the CH3 domain of the other heavy chain theamino acid Y at position 407 is substituted by T (numbering according toKabat EU index).

In one embodiment of a multispecific antibody as reported herein, whichis of IgG2 isotype, the approach described in WO 2011/090762 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody.

In one embodiment of a multispecific antibody as reported herein, theapproach described in WO 2009/089004 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said multispecificantibody as reported herein, in the CH3 domain of one heavy chain theamino acid K or N at position 392 is substituted by a negatively chargedamino acid (in one preferred embodiment by E or D, in one preferredembodiment by D), and in the CH3 domain of the other heavy chain theamino acid D at position 399 the amino acid E or D at position 356 orthe amino acid E at position 357 is substituted by a positively chargedamino acid (in one preferred embodiment K or R, in one preferredembodiment by K, in one preferred embodiment the amino acids atpositions 399 or 356 are substituted by K) (numbering according to KabatEU index). In one further embodiment, in addition to the aforementionedsubstitutions, in the CH3 domain of the one heavy chain the amino acid Kor R at position 409 is substituted by a negatively charged amino acid(in one preferred embodiment by E or D, in one preferred embodiment byD) (numbering according to Kabat EU index). In one even furtherembodiment, in addition to or alternatively to the aforementionedsubstitutions, in the CH3 domain of the one heavy chain the amino acid Kat position 439 and/or the amino acid K at position 370 is substitutedindependently from each other by a negatively charged amino acid (in onepreferred embodiment by E or D, in one preferred embodiment by D)(numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein, theapproach described in WO 2007/147901 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said multispecificantibody as reported herein, in the CH3 domain of one heavy chain theamino acid K at position 253 is substituted by E, the amino acid D atposition 282 is substituted by K and the amino acid K at position 322 issubstituted by D, and in the CH3 domain of the other heavy chain theamino acid D at position 239 is substituted by K, the amino acid E atposition 240 is substituted by K and the amino acid K at position 292 issubstituted by D (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein, theapproach described in WO 2007/110205 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody.

In one embodiment of all aspects and embodiments as reported herein themultispecific antibody is a bispecific antibody or a trispecificantibody. In one preferred embodiment of the invention the multispecificantibody is a bispecific antibody.

In one embodiment of all aspects as reported herein, the antibody is abivalent or trivalent antibody. In one embodiment the antibody is abivalent antibody.

In one embodiment of all aspects as reported herein, the multispecificantibody has a constant domain structure of an IgG type antibody. In onefurther embodiment of all aspects as reported herein, the multispecificantibody is characterized in that said multispecific antibody is ofhuman subclass IgG1, or of human subclass IgG1 with the mutations L234Aand L235A. In one further embodiment of all aspects as reported herein,the multispecific antibody is characterized in that said multispecificantibody is of human subclass IgG2. In one further embodiment of allaspects as reported herein, the multispecific antibody is characterizedin that said multispecific antibody is of human subclass IgG3. In onefurther embodiment of all aspects as reported herein, the multispecificantibody is characterized in that said multispecific antibody is ofhuman subclass IgG4 or, of human subclass IgG4 with the additionalmutation S228P. In one further embodiment of all aspects as reportedherein, the multispecific antibody is characterized in that saidmultispecific antibody is of human subclass IgG1 or human subclass IgG4.In one further embodiment of all aspects as reported herein, themultispecific antibody is characterized in that said multispecificantibody is of human subclass IgG1 with the mutations L234A and L235A(numbering according to Kabat EU index). In one further embodiment ofall aspects as reported herein, the multispecific antibody ischaracterized in that said multispecific antibody is of human subclassIgG1 with the mutations L234A, L235A and P329G (numbering according toKabat EU index). In one further embodiment of all aspects as reportedherein, the multispecific antibody is characterized in that saidmultispecific antibody is of human subclass IgG4 with the mutationsS228P and L235E (numbering according to Kabat EU index). In one furtherembodiment of all aspects as reported herein, the multispecific antibodyis characterized in that said multispecific antibody is of humansubclass IgG4 with the mutations S228P, L235E and P329G (numberingaccording to Kabat EU index).

In one embodiment of all aspects as reported herein, an antibodycomprising a heavy chain including a CH3 domain as specified herein,comprises an additional C-terminal glycine-lysine dipeptide (G446 andK447, numbering according to Kabat EU index). In one embodiment of allaspects as reported herein, an antibody comprising a heavy chainincluding a CH3 domain, as specified herein, comprises an additionalC-terminal glycine residue (G446, numbering according to Kabat EUindex).

In one embodiment the antibody comprises a first Fc-region polypeptideand a second Fc-region polypeptide, and

-   -   wherein    -   i) the first Fc-region polypeptide is a human IgG1 Fc-region        polypeptide and the second Fc-region polypeptide is a human IgG1        Fc-region polypeptide, or    -   ii) the first Fc-region polypeptide is a human IgG1 Fc-region        polypeptide with the mutations L234A, L235A and the second        Fc-region polypeptide is a human IgG1 Fc-region polypeptide with        the mutations L234A, L235A, or    -   iii) the first Fc-region polypeptide is a human IgG1 Fc-region        polypeptide with the mutations L234A, L235A, P329G and the        second Fc-region polypeptide is a human IgG1 Fc-region        polypeptide with the mutations L234A, L235A, P329G, or    -   iv) the first Fc-region polypeptide is a human IgG1 Fc-region        polypeptide with the mutations L234A, L235A, S354C, T366W and        the second Fc-region polypeptide is a human IgG1 Fc-region        polypeptide with the mutations L234A, L235A, Y349C, T366S,        L368A, Y407V, or    -   v) the first Fc-region polypeptide is a human IgG1 Fc-region        polypeptide with the mutations L234A, L235A, P329G, S354C, T366W        and the second Fc-region polypeptide is a human IgG1 Fc-region        polypeptide with the mutations L234A, L235A, P329G, Y349C,        T366S, L368A, Y407V, or    -   vi) the first Fc-region polypeptide is a human IgG4 Fc-region        polypeptide and the second Fc-region polypeptide is a human IgG4        Fc-region polypeptide, or    -   vii) the first Fc-region polypeptide is a human IgG4 Fc-region        polypeptide with the mutations S228P, L235E and the second        Fc-region polypeptide is a human IgG4 Fc-region polypeptide with        the mutations S228P, L235E, or    -   viii) the first Fc-region polypeptide is a human IgG4 Fc-region        polypeptide with the mutations S228P, L235E, P329G and the        second Fc-region polypeptide is a human IgG4 Fc-region        polypeptide with the mutations S228P, L235E, P329G, or    -   ix) the first Fc-region polypeptide is a human IgG4 Fc-region        polypeptide with the mutations S228P, L235E, S354C, T366W and        the second Fc-region polypeptide is a human IgG4 Fc-region        polypeptide with the mutations S228P, L235E, Y349C, T366S,        L368A, Y407V, or    -   x) the first Fc-region polypeptide is a human IgG4 Fc-region        polypeptide with the mutations S228P, L235E, P329G, S354C, T366W        and the second Fc-region polypeptide is a human IgG4 Fc-region        polypeptide with the mutations S228P, L235E, P329G, Y349C,        T366S, L368A, Y407V.

In one embodiment the antibody comprises a first Fc-region polypeptideand a second Fc-region polypeptide, and

-   -   wherein the antibody comprises the combination of mutations        -   i) I253A, H310A, and H435A, or        -   ii) H310A, H433A, and Y436A, or        -   iii) L251D, L314D, and L432D, or        -   iv) combinations of i) to iii)    -   in the first Fc-region polypeptide and in the second Fc-region        polypeptide.

In one embodiment of all aspects as reported herein the antibody asreported herein is an effector silent antibody. In one embodiment of allaspects as reported herein the antibody is an effector silent antibodyand does not bind to human FcRn. In one preferred embodiment of allaspects as reported herein is the antibody of the human subclass IgG1and has the mutations L234A, L235A, P329G, I253A, H310A and H434A inboth heavy chains (numbering according to the Kabat index).

Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. The required nucleic acidmay encode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychains of the antibody). In a further embodiment, one or more vectors(e.g., expression vectors) comprising such nucleic acid are provided. Ina further embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an anti-[[PRO]] antibody is provided,wherein the method comprises culturing a host cell comprising a nucleicacid encoding the antibody, as provided above, under conditions suitablefor expression of the antibody, and optionally recovering the antibodyfrom the host cell (or host cell culture medium).

For recombinant production of an antibody, nucleic acid encoding anantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli.) After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gemgross, T. U., Nat. Biotech. 22 (2004) 1409-1414; and Li,H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

Specific Embodiments

-   1. Use of a limited digestion with a proteolytic enzyme of a    multispecific antibody for the analysis of the multispecific    antibody's light chain pairing.-   2. Use of a limited digestion with a proteolytic enzyme of a    multispecific antibody for the determination of light chain    mispairing in the multispecific antibody.-   3. Use of a limited digestion with a proteolytic enzyme of a    multispecific antibody produced by a recombinant mammalian cell for    the selection of a multispecific antibody producing mammalian cell.-   4. The use according to any one of embodiments 1 to 3, wherein the    proteolytic enzyme is selected from the group consisting of Lys-C,    Asp-N, Arg-C, Glu-C and chymotrypsin.-   5. The use according to embodiment 4, wherein the proteolytic enzyme    is Lys-C.-   6. The use according to any one of embodiments 1 to 5, wherein the    incubating is for 35 to 45 minutes.-   7. The use according to embodiment 6, wherein the incubating is for    about 40 minutes.-   8. The use according to any one of embodiments 1 to 7, wherein the    multispecific antibody that is incubated with the proteolytic enzyme    is a deglycosylated multispecific antibody.-   9. The use according to any one of embodiments 1 to 8, wherein the    weight ratio of antibody to enzyme is about 1:200.-   10. The use according to any one of embodiments 1 to 9, wherein the    multispecific antibody that is incubated with the proteolytic enzyme    has a concentration of from 200 to 600 μg/mL.-   11. The use according to any one of embodiments 1 to 10, wherein the    multispecific antibody is a monoclonal multispecific antibody.-   12. The use according to any one of embodiments 1 to 11, wherein the    multispecific antibody comprises at least two non-peptidically bound    light chains.-   13. The use according to any one of embodiments 1 to 11, wherein the    multispecific antibody comprises at least three non-peptidically    bound polypeptides.-   14. A method for the determination of the light chain pairing in a    multispecific antibody comprising the following steps:    -   a) incubating a sample comprising the multispecific antibody        with a proteolytic enzyme for a limited time,    -   b) identifying the mass of the fragments obtained by the limited        proteolytic digestion in step a) by mass spectrometry, and    -   c) determining from the results in step b) the light chain        pairing of the multispecific antibody.-   15. A method for the determination of light chain mispairing of a    multispecific antibody comprising the following steps:    -   a) incubating a sample comprising the multispecific antibody        with a proteolytic enzyme for a limited time,    -   b) identifying the mass of the fragments obtained by the limited        proteolytic digestion in step a) by mass spectrometry, and    -   c) determining from the results in step b) the light chain        pairing of the multispecific antibody and thereby determining        light chain mispairing.-   16. A method for the selection of a recombinant mammalian cell    producing a multispecific antibody comprising the following steps:    -   a) individually incubating a sample comprising a multispecific        antibody produced by a clonal population of a recombinant        mammalian cell of a multitude of recombinant mammalian cells all        cells of the multitude producing the same multispecific antibody        with a proteolytic enzyme for a limited time,    -   b) identifying the mass of the fragments obtained by the limited        proteolytic digestion in step a) by mass spectrometry,    -   c) determining from the results in step b) the presence of light        chain mispairing of the multispecific antibody for each clonal        cell population, and    -   d) selecting based on the results in step c) a recombinant cell        producing a multispecific antibody.-   17. The method according to any one of embodiments 14 to 16, wherein    the proteolytic enzyme is selected from the group consisting of    Lys-C, Asp-N, Arg-C, Glu-C and chymotrypsin.-   18. The method according to embodiment 17, wherein the proteolytic    enzyme is Lys-C.-   19. The method according to any one of embodiments 14 to 18, wherein    the incubating is for 35 to 45 minutes.-   20. The method according to embodiment 19, wherein the incubating is    for about 40 minutes.-   21. The method according to any one of embodiments 14 to 20, wherein    the multispecific antibody that is incubated with the proteolytic    enzyme is a deglycosylated multispecific antibody.-   22. The method according to any one of embodiments 14 to 21, wherein    the weight ratio of antibody to enzyme is about 1:200.-   23. The method according to any one of embodiments 14 to 22, wherein    the multispecific antibody that is incubated with the proteolytic    enzyme has a concentration of from 200 to 600 μg/mL.-   24. The method according to any one of embodiments 14 to 23, wherein    step b) is    -   b) desalting the incubation mixture of step a) and identifying        the mass of the fragments obtained by the limited proteolytic        digestion in step a) by mass spectrometry,-   25. The method according to any one of embodiments 14 to 24, wherein    the multispecific antibody is a monoclonal multispecific antibody.-   26. The method according to any one of embodiments 14 to 25, wherein    the multispecific antibody comprises at least two non-peptidically    bound light chains.-   27. The method according to any one of embodiments 14 to 25, wherein    the multispecific antibody comprises at least three non-peptidically    bound polypeptides.

DESCRIPTION OF THE FIGURES

FIG. 1A-1B Correctly assembled (1) and light-chain mispaired (2) formsof a bivalent bispecific antibody.

FIG. 2A-2E Correctly assembled (1) and some light-chain mispaired (2, 3,4, 5) forms of a tetravalent bispecific antibody.

FIG. 3 IE of plasmin digested bivalent bispecific antibody; light chain1 and heavy chain 1 form the first binding site and light chain 2 andheavy chain 2 form the second binding site; the individual pairs wouldbe expected at the following positions: A: light chain 2+heavy chainfragment 1, B: light chain 1+heavy chain 1, C: light chain 2+heavy chain2, D: light chain 1+heavy chain 2.

FIG. 4 IE of plasmin digested bivalent bispecific antibody; light chain1 and heavy chain 1 form the first binding site and light chain 2 andheavy chain 2 form the second binding site; the individual pairs wouldbe expected at the following positions: A: light chain 2+heavy chainfragment 1, B: light chain 1+heavy chain 1, C: light chain 2+heavy chain2, D: light chain 1+heavy chain 2.

FIG. 5 IE of limited Lys-C digested bivalent bispecific antibody; lightchain 1 and heavy chain 1 form the first binding site and light chain 2and heavy chain 2 form the second binding site; the individual pairswould be expected at the following positions: A: light chain 2+heavychain fragment 1, B: light chain 1+heavy chain 1, C: light chain 2+heavychain 2, D: light chain 1+heavy chain 2.

FIG. 6 IE of a tetravalent bispecific antibody; A: 3 times light chain1; B: correctly assemble antibody; C: 3 times light chain 2.

FIG. 7 MS-analysis for Protein A affinity chromatography flow-through ofproteolytic digested tetravalent bispecific antibody, ISCID of 0; A:correctly paired Fab, B: mispaired Fab.

FIG. 8 MS-analysis for Protein A affinity chromatography flow-through ofproteolytic digested tetravalent bispecific antibody, ISCID of 90; A:correctly paired Fab, B: mispaired Fab.

FIG. 9 MS-analysis for Protein A affinity chromatography eluate ofproteolytic digested tetravalent bispecific antibody, ISCID of 0; B:correctly paired Fc-Fab, B: mispaired Fc-Fab.

FIG. 10 MS-analysis for Protein A affinity chromatography eluate ofproteolytic digested tetravalent bispecific antibody, ISCID of 90; A:correctly paired Fc-Fab, B: mispaired Fc-Fab.

FIG. 11 EI of a pepsin digested tetravalent bispecific antibody.

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according tothe numbering systems according to Kabat (Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) as definedabove.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 600-1800 bp long gene segments, which wereflanked by singular restriction endonuclease cleavage sites, wereassembled by annealing and ligating oligonucleotides including PCRamplification and subsequently cloned via the indicated restrictionsites e.g. KpnI/SacI or AscI/Pacl into a pPCRScript (Stratagene) basedpGA4 cloning vector. The DNA sequences of the subcloned gene fragmentswere confirmed by DNA sequencing. Gene synthesis fragments were orderedaccording to given specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies, variants of expressionplasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F)cells based either on a cDNA organization with or without a CMV-Intron Apromoter or on a genomic organization with a CMV promoter were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a ß-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene was composed of thefollowing elements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   an immunoglobulin heavy chain signal sequence,    -   the human antibody chain (wildtype or with domain exchange)        either as cDNA or as genomic organization with the        immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the antibody chains as described below weregenerated by PCR and/or gene synthesis and assembled by knownrecombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences were verifiedby DNA sequencing. For transient transfections larger quantities of theplasmids were prepared by plasmid preparation from transformed E. colicultures (Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Multispecific antibodies were expressed by transient co-transfection ofthe respective expression plasmids in adherently growing HEK293-EBNA orin HEK29-F cells growing in suspension as described below.

Transient Transfections in HEK293-EBNA System

Multispecific antibodies were expressed by transient co-transfection ofthe respective expression plasmids (e.g. encoding the heavy and modifiedheavy chain, as well as the corresponding light and modified lightchain) in adherently growing HEK293-EBNA cells (human embryonic kidneycell line 293 expressing Epstein-Barr-Virus nuclear antigen; Americantype culture collection deposit number ATCC #CRL-10852, Lot. 959 218)cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco®)supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco®), 2 mML-Glutamine (Gibco®), and 250 μg/ml Geneticin (Gibco®). For transfectionFuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) was usedin a ratio of FuGENE™ reagent (μl) to DNA (μg) of 4:1 (ranging from 3:1to 6:1). Proteins were expressed from the respective plasmids using amolar ratio of (modified and wildtype) light chain and heavy chainencoding plasmids of 1:1 (equimolar) ranging from 1:2 to 2:1,respectively. Cells were fed at day 3 with L-Glutamine ad 4 mM, Glucose[Sigma] and NAA [Gibco®]. Multispecific antibody containing cell culturesupernatants were harvested from day 5 to 11 after transfection bycentrifugation and stored at −20° C. General information regarding therecombinant expression of human immunoglobulins in e.g. HEK293 cells isgiven in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Transient Transfections in HEK293-F System

Multispecific antibodies were generated by transient transfection withthe respective plasmids (e.g. encoding the heavy and modified heavychain, as well as the corresponding light and modified light chain)using the HEK293-F system (Invitrogen) according to the manufacturer'sinstruction. Briefly, HEK293-F cells (Invitrogen) growing in suspensioneither in a shake flask or in a stirred fermenter in serum-freeFreeStyle™ 293 expression medium (Invitrogen) were transfected with amix of the four expression plasmids and 293Fectin™ or fectin(Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seededat a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8%CO2. The day after the cells were transfected at a cell density of ca.1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen)with 600 μg total plasmid DNA (1 μg/mL) encoding the heavy or modifiedheavy chain, respectively and the corresponding light chain in anequimolar ratio and B) 20 ml Opti-MEM+1.2 mL 293 fectin or fectin (2μl/mL). According to the glucose consumption glucose solution was addedduring the course of the fermentation. The supernatant containing thesecreted antibody was harvested after 5-10 days and antibodies wereeither directly purified from the supernatant or the supernatant wasfrozen and stored.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads were washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant was applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 hour at room temperature the beads were washed on anUltrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody was eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample was combined with NuPAGE® Sample Reducing Agent or leftunreduced, respectively, and heated for 10 min at 70° C. Consequently,5-30 μl were applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen)(with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE®Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE)and stained with Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants was quantitatively measured by affinity HPLCchromatography. Briefly, cell culture supernatants containing antibodiesand derivatives that bind to Protein A were applied to an AppliedBiosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH2.5 on an Agilent HPLC 1100 system. The eluted protein was quantified byUV absorbance and integration of peak areas. A purified standard IgG1antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Streptavidin A-96 well microtiter plates (Roche)are coated with 100 μL/well biotinylated anti-human IgG capture moleculeF(ab′)2<h-Fcγ>BI (Dianova) at 0.1 μg/mL for 1 hour at room temperatureor alternatively overnight at 4° C. and subsequently washed three timeswith 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/well of adilution series in PBS (Sigma) of the respective antibody containingcell culture supernatants was added to the wells and incubated for 1-2hour on a microtiterplate shaker at room temperature. The wells werewashed three times with 200 μL/well PBST and bound antibody was detectedwith 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detectionantibody for 1-2 hours on a micro-titerplate shaker at room temperature.Unbound detection antibody was washed away three times with 200 μL/wellPBST and the bound detection antibody was detected by addition of 100 μLABTS/well. Determination of absorbance was performed on a Tecan FluorSpectrometer at a measurement wavelength of 405 nm (reference wavelength492 nm).

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Example 1

Method for the Determination of Light Chain Mispairing of aMultispecific Antibody after Limited Proteolytic Digestion

The expected primary structures were analyzed by electrospray ionizationmass spectrometry (ESI-MS) of the limited LysC digested CrossMabs.Advantageously the antibody has been deglycosylated in advance.

The VH/VL CrossMabs was deglycosylated with N-Glycosidase F in aphosphate or Tris or histidine buffer at 37° C. for up to 17 h at aprotein concentration of 1 mg/mL and an antibody: enzyme ratio of 100:1.The limited Lys-C (Roche Diagnostics GmbH, Mannheim, Germany) digestionswas performed with 100 μg deglycosylated VH/VL CrossMabs in a Trisbuffer pH 8 at 37° C. for 40 min.

Prior to mass spectrometry the samples were desalted via HPLC on aSephadex G25 column (GE Healthcare).

The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MSsystem (Bruker Daltonik) equipped with a TriVersa NanoMate source(Advion).

Example 2

Method for the Determination of Light Chain Mispairing of a TetravalentBispecific Antibody

The tetravalent bispecific antibody was deglycosylated withN-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. forup to 17 h at a protein concentration of 1 mg/ml and an antibody:enzymeratio of 100:1. The limited Lys-C(Roche Diagnostics GmbH, Mannheim,Germany) digestions was performed with 100 μg deglycosylated tetravalentbispecific antibody in a 100 mM citrate buffer pH 3.7 at 37° C. for 16hours at a protein concentration of approx. 0.9 mg/mL and anantibody:enzyme ratio of 50:1.

What is claimed is:
 1. A method for determining light chain pairing in amultispecific antibody comprising the following steps: a) incubating asample comprising the multispecific antibody with a proteolytic enzymefor 35 to 45 minutes, wherein the proteolytic enzyme is Lys-C to producefragments of the multispecific antibody, b) identifying the mass of thefragments of the multispecific antibody obtained in step a) by massspectrometry, and c) determining from the mass of the fragmentsidentified in step b) the light chain pairing of the multispecificantibody, thereby determining light chain pairing of the multispecificantibody.
 2. The method according to claim 1, wherein the incubating isfor 40 minutes.
 3. The method according to claim 1, wherein themultispecific antibody that is incubated with the proteolytic enzyme isa deglycosylated multispecific antibody.
 4. The method according toclaim 1, wherein the weight ratio of antibody to enzyme is 1:200.
 5. Themethod according to claim 1, wherein the multispecific antibody that isincubated with the proteolytic enzyme has a concentration of from 200 to600 μg/mL.
 6. The method according to claim 1, wherein step b) furthercomprises: b) desalting the incubation mixture of step a) andidentifying the mass of the fragments obtained by the proteolyticdigestion in step a) by mass spectrometry.
 7. The method according toclaim 1, wherein the multispecific antibody is a monoclonalmultispecific antibody.
 8. The method according to claim 1, wherein themultispecific antibody comprises at least two non-peptidically boundlight chains.
 9. The method according to claim 1, wherein themultispecific antibody comprises at least three non-peptidically boundpolypeptides.
 10. A method for determining light chain mispairing in amultispecific antibody comprising the following steps: a) incubating asample comprising the multispecific antibody with a proteolytic enzymefor 35 to 45 minutes, wherein the proteolytic enzyme is Lys-C to producefragments of the multispecific antibody, b) identifying the mass of thefragments of the multispecific antibody obtained in step a) by massspectrometry, and c) determining from the mass of the fragmentsidentified in step b) the light chain pairing of the multispecificantibody, and thereby determining light chain mispairing of themultispecific antibody.
 11. The method according to claim 10, whereinthe incubating is for 40 minutes.
 12. The method according to claim 10,wherein the multispecific antibody that is incubated with theproteolytic enzyme is a deglycosylated multispecific antibody.
 13. Themethod according to claim 10, wherein the multispecific antibody that isincubated with the proteolytic enzyme has a concentration of from 200 to600 μg/mL.
 14. The method according to claim 10, wherein step b) furthercomprises: b) desalting the incubation mixture of step a) andidentifying the mass of the fragments obtained by the proteolyticdigestion in step a) by mass spectrometry.
 15. The method according toclaim 10, wherein the multispecific antibody is a monoclonalmultispecific antibody.
 16. The method according to claim 10, whereinthe multispecific antibody comprises at least two non-peptidically boundlight chains.
 17. The method according to claim 10, wherein themultispecific antibody comprises at least three non-peptidically boundpolypeptides.